Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end the specification, preceding the claims.
Genetic linkage studies have identified a number of loci associated with familial Alzheimer""s disease (1). Two of these loci encode related multipass transmembrane proteins, presenilins 1 and 2 (PS1 and PS2). Mutations in the genes encoding PS1 and PS2 loci are dominant and fully penetrant for early onset Alzheimer""s disease (2, 3, 4). The presenilins are ubiquitously expressed (3, 4), and found in conjunction with intracellular membranes (5). However, the normal role of presenilins, and the mechanism by which mutant presenilins cause Alzheimer""s disease, are not known.
Genetic studies in simple organisms offer a powerful approach to understanding the normal role of presenilins. The C. elegans sel-12 gene encodes a protein that displays about 50% amino acid sequence identity to PS1 and PS2 (6). The sel-12 gene was also described in the Patent Cooperation Treaty international application, PCT/US96/15727, filed Sep. 27, 1996 and U.S. Provisional Application No. 60/004,387, filed Sep. 27, 1995, the content of which have incorporated into this application by reference. Genetic analysis established that reducing or eliminating sel-12 activity causes an egg-laying defective (Egl) phenotype, and that sel-12 activity facilitates the activity of LIN-12 and GLP-1, two receptors of the LIN-12/Notch family (6). SEL-12 appears to be a bona fide presenilin, since human PS1 and PS2 can rescue the Egl phenotype of a sel-12 mutant (7). Furthermore, the membrane topology of SEL-12 and PS1 appears to be similar (8, 9, 10). In addition to the functional and structural similarities, expression studies indicate that SEL-12 and human presenilins are expressed throughout development in many different cell types (3, 4, 7).
In the original application (U.S. Provisional Application No. 60/004,387, filed Sep. 27, 1995), we stated that we would identify other C. elegans genes with sequence similar to sel-12. We have identified another candidate C. elegans presenilin based on predicted amino acid sequence by searching the genomic sequence database (11, 12). Here, we show that this gene, which we have named hop-1 [hop=homolog of presenilin], encodes a functional presenilin by demonstrating that HOP-1 can rescue the Egl defect of a sel-12 mutant. We also show that HOP-1 has characteristic features of presenilin membrane topology. Finally, we show that reducing hop-1 activity in a sel-12 mutant background results in novel phenotypes, suggesting that hop-1 and sel-12 are functionally redundant.
This invention provides an isolated nucleic acid molecule encoding a HOP-1 or a mutated HOP-1. In an embodiment, the isolated nucleic acid molecule is a DNA, cDNA, genomic DNA, synthetic DNA or RNA.
This invention further provides nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with the sequence of a nucleic acid molecule which encodes a HOP-1 or a mutated HOP-1.
The above an isolated nucleic acid molecule may be operatively linked to a promoter of RNA transcription. This invention also provide a vector which comprises the above described isolated nucleic acid molecule.
This invention also provides a host vector system for the production of a HOP-1 which comprises the vector having the above described isolated nucleic acid molecule and a suitable host.
This invention also provides a method for producing a polypeptide (e.g. HOP-1) which comprises growing a host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced.
This invention also provides a purified wild-type HOP-1 protein or purified wild-type HOP-1 fragment thereof.
This invention further provides a purified mutated HOP-1 protein or purified mutated HOP-1 fragment thereof. Further, this invention also provides a polypeptide comprising the amino acid sequence of HOP-1, including, but limited to, fusion proteins having part of their amino acid sequence of the amino acid sequence of HOP-1.
This invention provides a method for identification of a compound which is capable of binding specifically to the wild-type HOP-1 or mutated HIP-1 comprising contacting the compound with the purified HOP-1 protein of claim 11 or purified mutated HOP-1 protein under conditions permitting specific binding of said HOP-1 protein and the compound. In an embodiment, the compound is not previously known. This invention also provides a compound identified by the above method.
This invention provides an antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1. In an embodiment, the antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1 is a monoclonal antibody.
This invention also provides a method for production of an antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1 comprising steps of: a) administering an amount of the purified protein or fragment of wild-type HOP-1 or mutated HOP-1 to a suitable animal effective to produce an antibody against wild-type HOP-1 or mutated HOP-1 protein in the animal; and b) testing the produced antibody for capability to bind wild-type HOP-1 or mutated HOP-1.
This invention also provides a method for production of an antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1 comprising steps of: a) determining conserved regions revealed by alignment of the wild-type HOP-1 or mutated HOP-1 protein sequences; b) synthesizing peptides corresponding to the revealed conserved regions; c) administering an amount of the synthesized peptides to a suitable animal effective to produce an antibody against the peptides in the animal; and d) testing the produced antibody for capability to bind wild-type HOP-1 or mutant HOP-1.
This invention also provides a method for production of an antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1 by in vitro immunization.
This invention also provides a method for production of an antibody capable of specifically binding to wild-type HOP-1 or mutated HOP-1 by in vitro immunization. Further, the antibody may be labeled.
This invention provides transgenic animal comprising the isolated nucleic molecule encoding a HOP-1 or a mutated HOP-1, specifically the transgenic animal is a Caenorhabditis elegans. 
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising administering an effective amount of the compound to the above transgenic animal, alteration of the conditions of the transgenic animal indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) treating Caenorhabditis elegans mutants having reduced, increased or altered HOP-1 activity with the compound; and b) determining whether the compound suppresses, enhances or has no effect on the phenotype of the mutants, the suppression or enhancement of the phenotype indicating the compound is capable of ameliorating Alzheimer""s disease.
This invention also provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) contacting the compound with a cell which expresses HOP-1; and b) determining whether the compound increases, decreases or has no effect on the amount of HOP-1 level or activity in the cell, the increase or decrease of HOP-1 indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising administering an effective amount of the compound to the transgenic animal expressing exogenous HOP-1 and SEL-12, alteration of the conditions of the transgenic animal indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) treating Caenorhabditis elegans mutants having reduced, increased or altered HOP-1 and SEL-12 activity with the compound; and b) determining whether the compound suppresses, enhances or has no effect on the phenotype of the mutants, the suppression or enhancement of the phenotype indicating the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) contacting the compound with a cell which expresses both HOP-1 and SEL-12; and b) determining whether the compound increases, decreases or has no effect on the amount of SEL-12 level or activity or HOP-1 level or activity in the cell, the increase or decrease of SEL-12 or HOP-1 indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention also provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising administering an effective amount of the compound to the transgenic animal which expresses exogenous HOP-1 and LIN-12, alteration of the conditions of the transgenic animal indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) treating Caenorhabditis elegans mutants having reduced, increased or altered HOP-1 and LIN-12 activity with the compound; and b) determining whether the compound suppresses, enhances or has no effect on the phenotype of the mutants, the suppression or enhancement of the phenotype indicating the compound is capable of ameliorating Alzheimer""s disease.
This invention provides method for identifying a compound which is capable of ameliorating Alzheimer""s disease comprising: a) contacting the compound with a cell which expresses both HOP-1 and LIN-12; and b) determining whether the compound increases, decreases or has no effect on the amount of LIN-12 level or activity or HOP-1 level or activity in the cell, the increase or decrease of LIN-12 or HOP-1 indicating that the compound is capable of ameliorating Alzheimer""s disease.
This invention provides a method for producing suppressors of a hop-1 allele comprising: a) mutagenizing hop-1; sel-12 mutant hermaphrodites with an effective amount of a mutagen; b) screening for revertants in the F1, F2, and F3 generations; and c) isolating the screened revertants. This invention also provides for a suppressor identified by the above-described method.
This invention provides a method for identifying a suppressor gene comprising performing DNA sequence analysis of the above suppressor to identify the suppressor gene. This invention also provides an enhancer identified by the above-described method.
This invention provides a method for producing enhancers of a hop-1 allele comprising: a) mutagenizing hop-1 mutant hermaphrodites with an effective amount of a mutagen; b) screening for enhanced mutants or synthetic phenotypes in the F1, F2, and F3 generations; and c) isolating the enhanced mutants. This invention also provides for a suppressor identified by the above-described method.
This invention provides a method for identifying an enhancer gene comprising performing DNA sequence analysis of the above enhancer to identify the enhancer gene. This invention also provides an enhancer identified by the above-described method.
This invention provides a pharmaceutical composition comprising an effective amount of the compound identified by the above methods and a pharmaceutically acceptable carrier.
This invention provides a method of ameliorating Alzheimer""s disease which comprises administrating the above pharmaceutical composition in an amount effective to ameliorate Alzheimer""s disease.